Many natural physiological functions such as tissue remodeling, inflammation, coagulation, and fibrinolysis require proteolytic enzymes. Of particular importance is a mechanistic class of proteases called serine proteases. The active site of all functional members of the serine protease family contains a characteristic catalytic triad consisting of serine (hence the name), aspartic acid and histidine. The hydroxyl group of the catalytic site serine is involved in a nucleophilic attack on the carbonyl carbon of the peptide bond to be hydrolyzed resulting in acylation of the protease and hydrolysis of the peptide bond. This is followed rapidly by a deacylation step resulting in the release of intact protease.
Although originally named for their mechanism of action, members of the serine protease family also show significant sequence and structural homology. Some serine proteases are very specific, cleaving only certain peptide bonds of a specific target protein while others are very nonspecific, degrading multiple target proteins into small peptides.
Serine proteases are regulated at many levels. Some are synthesized as inactive proenzymes and are activated only during specific events and at specific locations. This allows the body to respond rapidly to a physiological perturbation by activating an already present reservoir of proteolytic activity. Coagulation, for example, is carried out when circulating proenzymes such as factor X and prothrombin are sequentially activated in response to injury resulting in a cascade of clotting activity. In addition, proteolytic activity is often localized to specific sites, such as receptor binding sites which can cause high local concentrations of protease or proenzyme ready for activation.
Once activated, it is extremely important that proteolytic activity be confined both spatially and temporally. This control is often achieved by the presence of specific inhibitors which block proteolytic activity. An important family of related proteins, the serine protease inhibitors, or "serpins", are key in the regulation of serine proteases. Like the serine proteases, serpins were first defined by their common mechanism of action but later turned out to be highly homologous both in terms of sequence and structure.
Serpins all contain an inhibitor domain with a reactive peptide bond defined on either side by the P.sub.1 and P.sub.1 ' in a direction to the left away from the reactive site, the amino acids are referred to as P.sub.1, P.sub.2, P.sub.3, etc., and in a direction to the right away from the reactive site they are referred to as P.sub.1 ', P.sub.2 ', P.sub.3 ', etc. The P.sub.1 residue is recognized by the substrate binding pocket of the target protease which attacks the reactive peptide bond as though a normal substrate. However, hydrolysis of the peptide bond and release of the protease does not proceed to completion. The normal deacylation step is so slow that the reaction becomes essentially irreversible and the protease becomes trapped in a stable, equimolar complex.
Protease nexin-I (PN-I) is a member of the serpin family. PN-I is produced by many different cell types including fibroblasts, glial cells, and platelets. PN-I is secreted by cells into the extracellular environment where it binds to and inhibits target serine proteases. PN-I-protease complexes then bind back to specific cell surface receptors where they are internalized and degraded.
PN-I is very similar, both structurally and functionally to antithrombin (AT-III). AT-III is the primary plasma inhibitor of blood coagulation. The inhibition of thrombin by AT-III in plasma is normally very weak but is accelerated significantly by the presence of heparin or by other mucopolysaccharides on the endothelial lining of blood vessels. The therapeutic value of heparin as a blood "thinning" agent is due to its enhancement of AT-III activity. Like AT-III, PN-I has a high affinity heparin binding site and inhibits thrombin much more rapidly (50-100 fold) in the presence of heparin. Thus PN-I has therapeutic potential as an anticoagulant.
On the other hand, PN-I differs from AT-III in a number of ways. Unlike AT-III, PN-I is also a good inhibitor of the fibrinolytic enzymes urokinase and plasmin, as well as trypsin. Furthermore, PN-I is not found in significant quantities in plasma and may function primarily in the tissues. The high affinity heparin binding site of PN-I serves to localize it to connective tissues and cells which contain sulfated proteoglycans on their surface and surrounding extracellular matrix. Thus PN-I's primary role seems to be in regulating proteolytic activity in tissues as opposed to blood. Further evidence for the role of PN-I is found by the fact that it is present in brain tissue and may be involved in peripheral nerve regeneration and neurite extension.
The relative efficiency with which PN-I inhibits serine proteases can be measured by the second order association rate constant (k.sub.ass) as previously described in Bieth, J. G. (Bull. Euro. Physiopath. Resp. (1980) 16:183-195), and reported by Scott et al. (J. Biol. Chem. (1985) 260:7029-7034), both of which are incorporated herein by reference to disclose and explain the meaning of the rate association constant. In general, a value for k.sub.ass equal to or greater than 1.times.10.sub.5 M.sub.-1 S.sub.-1 for a particular protease-inhibitor reaction is considered to be physiologically significant (Travis and Salveson Ann. Rev. Biochem. (1983) 52:655-709). The k.sub.ass or rate association constant has inverse-mole-seconds as its units, and the larger the k.sub.ass, the more rapid the inhibition. Accordingly, a k.sub.ass value is always given as a value with respect to a particular enzyme and is zero if there is no inhibition of the enzyme.
Many physiologically important protease inhibitor reactions such as elastase-alpha-1 antitrypsin and plasmin-alpha-2-antiplasmin occur with rate constants as high as 1.times.10.sup.7 M.sup.-1 S.sup.-1 or greater. The thrombin-PN-I reaction occurs at a similar high rate in the presence of heparin.
Protease nexin I (PN-I) has been purified from serum-free medium conditioned by human foreskin cells (Scott, R. W. et al., J Biol Chem (1983) 58:1043910444). It is a 43 kd glycoprotein which is released by fibroblasts, myotubes, heart muscle cells, and vascular smooth muscle cells. Its release, along with that of plasminogen activator, is stimulated by phorbol esters and by mitogens (Eaton, D. L. et al., J cell Biol (1983) 123:128). Native PN-I is an approximately 400 amino acid protein containing about 10% carbohydrate. Since it is present only in trace levels in serum, it apparently functions at or near the surfaces of interstitial cells. PN-I inhibits all the known activators of urokinase proenzyme, plasmin, trypsin, thrombin, and factor Xa (Eaton, D. L. et al., J Biol Chem (1984) 259:6241). It also inhibits tissue plasminogen activator and urokinase. However, PN-I does not inhibit elastase.
Because the reactive site region of PN-I acts as a substrate analogue the present inventors postulated that it might be possible to drastically alter PN-I activity by modifying the reactive site sequence of PN-I, thus changing its protease specificity. Similar efforts with alpha-1-antitrypsin, for example, resulted in variants with altered and therapeutic potential (M. Courtney et al., Nature (1985) 313:149-151). PN-I is different from most serpins in that it is found in tissues, contains a high affinity heparin binding site which localizes it to tissues, and has a tissue clearance receptor that is responsible for endocytosis of protease-PN-I complexes. Thus the present inventor further postulated that it might be possible to generate PN-I variants as inhibitors of physiologic proteases such as elastase which, if possible, could result in molecules with very unique therapeutic potential for connective tissue diseases.